The carbon metabolism of newly isolated Clostridium tyrobutyricum JM1 was investigated at varying initial glucose concentrations (27.8-333.6mM). Because an understanding of metabolic regulations was required to provide guidance for further effective metabolic design or optimization, in this case, maximizing hydrogen production, carbon and energy balances by C. tyrobutyricum JM1 were determined and applied in anaerobic glucose metabolism. The overall carbon distribution suggested that initial glucose concentrations had strong influence on the stoichiometric coefficients of products and the molar production of ATP on the formation of biomass. C. tyrobutyricum JM1 had a high capacity for hydrogen production at the initial glucose concentration of 222.4 mM with high concentrations of acetate and butyrate.